Survival in rectal cancer is predicted by T cell infiltration of tumour-associated lymphoid nodules.

Lymphoid nodules are a normal component of the mucosa of the rectum, but little is known about their function and whether they contribute to the host immune response in malignancy. In rectal cancer specimens from patients with local (n=18), regional (n=12) and distant (n=10) disease, we quantified T cell (CD3, CD25) and dendritic cell (CD1a, CD83) levels at the tumour margin as well as within tumour-associated lymphoid nodules. In normal tissue CD3+, but not CD25+, T cells are concentrated at high levels within lymphoid nodules, with significantly fewer cells found in surrounding normal mucosa (P=0.001). Mature (CD83), but not immature (CD1a), dendritic cells in normal tissue are also found clustered almost exclusively within lymphoid nodules (P=<0.0001). In rectal tumours, both CD3+ T cells (P=0.004) and CD83+ dendritic cells (P=0.0001) are also localized preferentially within tumour-associated lymphoid nodules. However, when comparing tumour specimens to normal rectal tissue, the average density of CD3+ T cells (P=0.0005) and CD83+ dendritic cells (P=0.0006) in tumour-associated lymphoid nodules was significantly less than that seen in lymphoid nodules in normal mucosa. Interestingly, regardless of where quantified, T cell and dendritic cell levels did not depend upon the stage of disease. Increased CD3+ T cell infiltration of tumour-associated lymphoid nodules predicted improved survival, independent of stage (P=0.05). Other T cell (CD25) markers and different levels of CD1a+ or CD83+ dendritic cells did not predict survival. Tumour-associated lymphoid nodules, enriched in dendritic cells and T cells, may be an important site for antigen presentation and increased T cell infiltration may be a marker for improved survival.

The importance of MUC1 cellular localization in patients with breast carcinoma: an immunohistologic study of 71 patients and review of the literature.

BACKGROUND: The MUC1 mucin is present on the apical surface of normal secretory epithelia. In breast carcinoma, MUC1 expression is variable in amount and cellular localization, the significance of which is controversial. The authors undertook a detailed analysis of staining pattern combined with a comprehensive literature review to better understand the role of MUC1 in breast carcinoma. METHODS: Seventy-one patients with breast carcinoma were examined for MUC1, beta-catenin, and E-cadherin staining patterns. These data were compared with data from 25 articles from the literature examining the expression of MUC1 in breast carcinoma. RESULTS: All invasive carcinomas showed some MUC1 staining. In invasive ductal carcinomas, MUC1 was detected in the apical membrane (15%), cytoplasm (93%), or circumferential membrane (13%), with 81% of tumors showing a mixture of patterns. Tumors with low overall MUC1 expression (< or = 50% positive tumor cells) had a higher nuclear grade than tumors with high overall MUC1 expression (> 50%; P = 0.01). Tumors with high and low cytoplasmic expression had no difference in nuclear grade (P > 0.3). Circumferential membrane staining was correlated with positive lymph node status (P = 0.011). CONCLUSIONS: In the literature, similar findings prevailed in which overall MUC1 expression was increased in lower grade (10 of 14 studies), estrogen receptor positive (8 of 13 studies) tumors and was associated with a better prognosis (8 of 13 studies). High cytoplasmic staining was associated with a worse prognosis, an association that was not explained by differences in histologic grade. Thus, the presence of MUC1 in the majority of tumor cells is associated with better differentiated tumors and with an improved prognosis. However, aberrantly localized MUC1 in the tumor cell cytoplasm or nonapical membrane is associated with a worse prognosis.

Effect of radiation on cytokine and cytokine receptor messenger-RNA profiles in p53 wild and mutated human glioblastoma cell lines.

OBJECTIVE: Glioblastoma cells produce cytokines with proinflammatory or immunosuppressive properties, or both, which, in addition to altered p53 gene expression, have been shown to be associated with glioblastoma resistance to radiotherapy. The reported data concerning cytokines have been isolated and sometimes discordant, and a comprehensive profile analysis of cytokines and their corresponding receptors in irradiated glioblastomas has received limited attention. The object of this study was to test the hypothesis that radiation alone in clinically relevant doses would not significantly alter expression of endogenous cytokines and their receptors in human glioblastoma celll ines with wild-type and mutant p53. DESIGN AND METHOD: Culture specimens of 4 glioblastoma cell lines of different p53 gene expression (U87, U118, U251, U373) were irradiated with cobalt 60 at a dose of 10 Gy. After 48 hours, radiosensitivity was defined through a colony formation assay, cell cycle distribution was analyzed by flow cytometry, and cytokine and cytokine receptor messenger-RNA (mRNA) profiles were defined with an RNase protection assay. Different single doses of radiation at varying time intervals after culture were applied also to wild-type p53 cell lines. RESULTS: All cell lines were relatively radioresistant at lower doses of 1 and 2 Gy. Immunosuppressive cytokine and cytokine receptor mRNA of the Th2 (IL-13Ralpha, IL-4) and Th3 family (TGF-beta1, 2 and 3, TGF-betaRI and RII) were expressed. In contrast, only 2 proinflammatory Th1 cytokine receptor genes (IFN-gammaRa and IFN-gammaRbeta), but no significant Th1 cytokine gene expression, were detected. Even though the population examined included a large fraction of reproductively dead cells, cytokine and cytokine receptor mRNA profiles were not altered significantly by irradiation in all cell lines, regardless of the p53 status. CONCLUSION: These results suggest that cobalt irradiation alone at clinically relevant doses does not significantly alter the cytokine and cytokine receptor profiles in human glioblastoma cell lines.

Sinusoidal CD30-positive large B-cell lymphoma: a morphologic mimic of anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) has been recognized recently as a distinct clinicopathologic entity, restricted to a subset of CD30-positive diffuse large cell lymphomas of T/null lineage. Some of the characteristic features of ALCL, such as CD30 antigen expression and the presence of large pleomorphic lymphoid cells infiltrating lymph node sinuses, can be found rarely in diffuse large B-cell lymphomas. We collected 11 such cases, and their clinical, morphologic, and immunophenotypic features are reviewed. The age of the patients ranged from 36 to 82 years (mean, 63.2 years) with a male to female ratio of 1:1.2. All neoplasms were nodal with a sinusoidal infiltrative pattern, although four neoplasms also had foci of confluent growth. Eight tumors were composed predominantly of large pleomorphic cells with occasional Reed-Sternberg-like cells. The other three tumors had a higher proportion of large monomorphic lymphoid cells. Necrosis and admixed granulocytes were other common features. Immunophenotypically, all cases were positive for CD30 and CD20 or CD79a. All eight cases examined for anaplastic lymphoma kinase-1 immunoreactivity were negative. In situ hybridization for Epstein-Barr virus RNA was performed in eight cases; two were positive. Excluding one consultation case with no available clinical follow-up data, six patients died of the disease within 3 years and one had disease relapse within 1 year. We conclude that an unusual variant of diffuse large B-cell lymphoma can closely mimic ALCL. However, these neoplasms can be distinguished from ALCL by virtue of their B-lineage and lack of anaplastic lymphoma kinase-1 expression. Evidence of Epstein-Barr virus infection can be found in a small subset of these neoplasms.

Hepatosplenic T-cell lymphoma of alphabeta lineage in a 16-year-old boy presenting with hemolytic anemia and thrombocytopenia.

The authors report an unusual case of peripheral T-cell lymphoma in a 16-year-old boy who presented initially with jaundice, splenomegaly, anemia, and thrombocytopenia. A lymphoma was found subsequently in the spleen, which was infiltrated extensively in the red pulp by medium-sized, blastic-appearing lymphoma cells. Immunologic characterization of these cells revealed positivity for CD3, CD5, CD45RO, CD56, and T-cell intracellular antigen (TIA), and negativity for CD2, CD3, CD4, CD8, CD57, CD34, and terminal deoxynucleotidyl transferase (TdT). Conventional cytogenetic studies revealed the presence of isochromosome 7q. On follow up, this patient deteriorated rapidly, with evidence of liver and bone marrow involvement. Although the overall clinical and pathologic features of this disease were characteristic of hepatosplenic gammadelta T-cell lymphoma, the T-cell receptor of this tumor showed an immunophenotype of alphabeta not gammadelta lineage. Using the Southern blot technique, the authors demonstrated monoclonal gene rearrangement of the T-cell receptor beta-chain. Thus, they confirmed the existence of hepatosplenic alphabeta T-cell lymphoma. In view of its overall similarity to hepatosplenic gammadelta T-cell lymphoma, this unusual entity probably represents a slight biologic variation of the same disease.

Expression of transmembrane 4 superfamily member, CD9, is related to improved progression-free survival in patients with diffuse non-Hodgkin's lymphoma.

Decreased expression of the transmembrane 4 superfamily member, CD9, is associated with poor prognosis in patients with breast or non-small cell lung cancer. The expression of CD9 in lymphoma was examined in this study. Fifty-one sections with diffuse lymphomas were examined. Thirty-seven had low expression and 14 high expression of CD9. At 5 years the progression-free survival rates were 83.3+/-10.8% and 32.8+/-9.2% (p=0.018), and the actual survival were 83.3+/-10.8% and 56.8+/-8.9% (p=0.256) for those with high and low CD9 expression respectively. Decreased expression of CD9 appears to be a prognostic factor for poor survival in patients with diffuse lymphomas.

Ex vivo fine-needle aspiration cytology and flow cytometric phenotyping in the diagnosis of lymphoproliferative disorders: a proposed algorithm for maximum resource utilization.

Surface marker characterization of lymphoproliferative disorders is an essential component in the diagnostic work-up of these lesions. Immunohistochemical surface marker analysis (SMA) is somewhat costly, fixation-dependent, and difficult to objectively quantitate. Two-color flow cytometric (TCFCM) SMA allows for more quantitative dual marker analysis of a wide range of surface antigens, and is less expensive. Ex vivo fine-needle aspiration (xvFNA) has been reliably used for FCM DNA analysis. The procedure has also been used to harvest tumor cells for xenotransplantation. In this study, we attempted to test the reliability of material obtained by xvFNA for SMA. We also designed an algorithm initiated by cytological assessment of the xvFNA smears in order to tailor the panel of antibodies required for TCFCM SMA of the aspirates. We performed 20 xvFNAs on freshly resected specimens from 19 patients with suspected lymphoproliferative disorders. The specimens included 12 lymph node biopsies, seven splenectomies, and one breast biopsy. There were 10 male and nine female patients with a median age of 58 yr. The aspirate cell suspensions were examined by FCM within 24 hr of harvesting. The number of markers used ranged from four to 14 with an average of eight. The diagnoses included non-Hodgkin's lymphoma (n = 5), lymphocytic leukemia (n = 5), reactive lymphoid hyperplasia (n = 8), and Hodgkin's disease (n = 1). Combining cytological assessment of the xvFNA smears and TCFCM SMA, the diagnosis was reached prior to histopathologic examination in 17 cases (90%). The two remaining cases showed a reactive pattern on cytology and a polyclonal FCM SMA profile, and the diagnosis of sarcoidosis and toxoplasmosis was made on histological examination. Our study suggests that xvFNA provides adequate material for TCFCM SMA. An algorithm combining xvFNA cytology, FCM SMA, and histological examination is appropriate for the diagnosis of lymphoproliferative disorders in most instances with maximal resource utilization and minimal expense.

Mutant p53 expression in kidney tubules adjacent to renal cell carcinoma: evidence of a precursor lesion.

Neoplastic transformation can be associated with mutations of the p53 gene. This leads to stabilization of its protein product and to its accumulation, which allows immunohistochemical detection. Mutant p53 expression has been seen in many neoplasms, including renal cell carcinoma (RCC). We recently described putative precursor lesions of RCC. The lesions were defined as intratubular epithelial dysplasia (IED) of kidney tubules adjacent to RCC. They were seen in one-third of the cases studied. The findings were based only on light microscopic analysis. We hypothesized that neoplastic transformation would be manifested by mutant p53 expression in the kidney tubules adjacent to RCC and not in nonneoplastic kidneys. Immunohistochemical staining for p53 in 24 cases of RCC with adjacent kidneys was performed. We used the DO-7 monoclonal antibody reactive for the N-terminal of the p53 protein on formalin-fixed paraffin-embedded tissue. Sections from 14 kidneys resected for nonneoplastic conditions were used as controls. Twenty-one (87%) of the 24 cases of RCC had nuclear p53 expression in the tumor cells. This included 14 cases (58%) with intense reactivity and 7 cases (29%) with weaker p53 immunoreactivity. Of the 24 cases of RCC, IED was identified in 13 cases (54%). Immunoreactivity for p53 was focally seen in tubules of all the lesions, as well as in the nonlesional areas. Six of the lesions exhibited intense nuclear staining. The kidneys adjacent to the RCC, with no evidence of IED, showed focally intense positive p53 nuclear staining in four cases. None of the control specimens showed p53 expression. Our findings provide supportive evidence that previously described IED in kidneys adjacent to RCC are most likely precursor lesions of the neoplasm. Aberrant expression of p53 in areas without evidence of IED may suggest that neoplastic transformation manifested by p53 mutation in kidney tubules may be seen before the development of the morphologic features of dysplasia and malignancy.

Localization of cytosolic aldehyde dehydrogenase in the developing chick retina: in situ hybridization and immunohistochemical analyses.

Cytosolic aldehyde dehydrogenase (ALDH) mRNA is present at high levels in the undifferentiated chick retina. Tissue maturation is accompanied by a 20-25x decrease in transcript levels. To determine the spatial and temporal distribution pattern of the ALDH transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development. The ALDH transcript and protein were detected at the earliest stage tested, in the inner layer of the optic cup of stage 14 (day 2) embryos. Both the ALDH transcript and protein were found in the dorsal retina of chick embryos from stage 18 (day 3) to day 16 of incubation. Accumulation of the ALDH protein in the neurites of ganglion cells could readily be detected at early developmental stages. Staining of this ganglion fiber layer was strong in the dorsal retina and could be followed up to and into the optic nerve. By day 11, ALDH mRNA was located primarily in the ciliary margin and in the inner nuclear layer of the dorsal retina. In addition to these areas, the ALDH protein was also found in the inner plexiform and optic nerve fiber layers. These results suggest that environmental or transcriptional factors involved in the regulation of the ALDH gene are restricted to the dorsal retina at early developmental stages and that there is a requirement for the compartmentalization of the ALDH transcript/protein in the undifferentiated chick retina.

Localization of a fatty acid binding protein and its transcript in the developing chick retina.

The undifferentiated chick retina has elevated levels of fatty acid binding protein (R-FABP) mRNA. Tissue maturation is accompanied by a 50-100-fold decrease in transcript levels. To determine the location of the R-FABP transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development. The R-FABP mRNA and protein were found throughout the retina from day 3 to day 7 of incubation. Accumulation of R-FABP in the neurites of ganglion cells could readily be detected at early developmental stages. By day 11, R-FABP transcript levels were considerably reduced in the retina, while the protein was primarily found in the inner nuclear layer, inner plexiform layer and optic nerve fiber layer of the retina. As well, R-FABP mRNA and protein were abundant in the non-pigmented ciliary epithelium, which represents the forward prolongation of the retina in the anterior eye. Immunoelectron microscopy revealed the presence of R-FABP in both the nucleus and cytoplasm of day 4 retinal cells. In the day 13 retina, R-FABP was abundant in the processes of neuronal cells. These results suggest that, early in retinal development, there is a requirement for FABP in the nucleus as well as the cytoplasm of all retinal cells. At later stages, the concentration of R-FABP in the processes of neuronal cells would suggest a biochemical or structural role related to neurite extension and synapse formation.

DNA analysis (ploidy) of molar pregnancies with image analysis on paraffin tissue sections.

Hydatidiform moles can be subclassified based on their ploidy. In general, complete moles are diploid, and partial moles are triploid. The standard method for the determination of DNA content is flow cytometric analysis. In this study, the authors investigated whether static cytometric analysis with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, IL) with a software program designed for quantitation of nuclear DNA content in tissue sections can be used to classify moles. Tissue sections from 17 moles were analyzed with this system, and the results were compared with those obtained with flow cytometric analysis. It was found that cell selection was an important factor. A high proportion of the hyperplastic trophoblast was in G2M. Exclusion of these areas and measurement of the trophoblast lining the villi only led to reliable results, and complete agreement between the results of the two methods was obtained. The findings indicate that cytometric analysis on tissue sections is a reliable alternative to flow cytometric analysis for the designation of moles as diploid or triploid.

Reactivity of monoclonal antibody B-ly7 with a subset of activated T cells and T-cell lymphomas.

Antibody B-ly7 is reactive with hairy cell leukemia and a small subpopulation of normal lymphocytes. The B-ly7 antigen can also be induced on normal peripheral blood lymphocytes by phorbol ester stimulation. Recently it has been found that the reactivity pattern of B-ly7 is similar to that of HML-1, an antibody reactive with mucosal T lymphocytes and so called enteropathy associated T-cell lymphomas. Reactivity of B-ly7 with 6/61 peripheral T-cell lymphomas, including two intestinal and four extraintestinal cases, is described. The intestinal cases were CD8 and CD7 positive, whereas the extraintestinal cases were CD4 positive and CD7 negative. Activation of purified peripheral blood T cells resulted in approximately 20% B-ly7+ T cells at Day 3. Approximately 75% of the B-ly7+ cells were CD8+, whereas the remainder were CD4+. The results indicate that B-ly7 as well as HML-1 recognize an activation-associated antigen that is expressed on small normal T-cell and B-cell populations and can be induced on a relatively high proportion of T and B cells in vitro.

Case study: application of precede and proceed as a framework for designing culturally sensitive diarrhea prevention programs and policy in arab countries.

This study utilized the diagnostic approach of the PRECEDE-PROCEED model. The constituents of this model were identified by utilizing participant observation field studies, traditionally applied by ethnographers to collect data describing the multiple facets of a culture, including linguistic patterns that capture and define characteristic semantics and viewpoints. The household study sample was randomly selected from a defined list of households known to have at least one child less than five years of age. An open-ended questionnaire format was used to interview the 115 mothers in the selected household sample. Kalama, the study community, is characteristically an agricultural village, situated in the Governorate of Kaliobia and located approximately 25 km (about 15.5 miles) from the capital, Cairo, Egypt. The latest 1986 census indicates a population size of about 13,328 people in 4,818 households. There were sixty-five deaths occurring among children less than five years in 1986. The causes of death were primarily related to diarrhea, followed by upper respiratory infections, congenital anomalies and birth injuries. This study outlines a) practices related to the management of diarrhea, including the administration of foods and drinks during such episodes; b) influences of governmental policies; and c) recommended strategies for overcoming barriers and promoting effective diarrhea intervention programs.

Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta.

Human trophoblast differentiates by the fusion of cytotrophoblasts to form syncytiotrophoblast. To determine factors controlling this process, the effects of epidermal growth factor (EGF) on trophoblast differentiation were studied using long term serum-free culture of isolated trophoblast. Only trophoblast was present in the cultures, as demonstrated by positive immunoperoxidase staining with beta hCG, cytokeratin, and trophoblast-specific H315 monoclonal antisera and by the absence of contaminating endothelial cells, fibroblasts, and macrophages, as shown by negative staining with vimentin and OKM1 monoclonal antisera. EGF induced large sustained increases in hCG and human placental lactogen (hPL) secretion in a dose-dependent manner. The minimum effective dose was 0.1 ng/mL, and the maximum effective dose was 1 ng/mL. Light and electron microscopic studies showed EGF-induced differentiation of cytotrophoblast to form syncytiotrophoblast. DNA content and cell number did not change during the process. The formation of syncytia thus probably accounted for the increase in hCG and hPL secretion. We conclude that EGF causes morphological differentiation, but not cell proliferation, of trophoblasts, and the differentiation results in increased hCG and hPL secretion from the syncytia.